Meier-Gorlin Syndrome (MGS) is a rare form of human primordial dwarfism characterized by a short stature, small external ears, and absent kneecaps. Recently, mutations in the Pre-Replication Complex (Pre-RC) were linked to MGS. The Pre-RC is composed of multiple proteins that must be present in order to initiate DNA replication. Although a genetic link has been made for MGS, it is unclear how these mutations cause the phenotypes observed in humans. To better understand the mutations’ phenotypes on a cellular and molecular level, our lab is utilizing the budding yeast, Saccharomyces cerevisiae. The MGS mutation in the subunit ORC4 of the Pre-RC occurs in a highly conserved region of the protein and an equivalent mutation has been made in budding yeast (orc4^MGS). Several interesting phenotypes have been observed in orc4^MGS haploid yeast including slow growth, a reduction in rDNA repeat number, and an altered replication program. However, it is unclear how the mutation is functioning to cause these phenotypes. The mutation could either be changing the protein’s function or causing the protein to become unstable and targeted for destruction, thus reducing ORC4 protein levels. To address this question, I am creating diploid strains either homozygous for orc4^MGS or heterozygous for orc4^MGS and orc4 deletion; to test the effect of lowering orc4^MGS dosage on growth, replication and rDNA repeat number. If the phenotypes are caused by the mutation’s function, the homozygous and heterozygous mutants phenotypes should remain similar. Conversely, if the mutation causes protein instability, and therefore a reduction of protein available for the Pre-RC, the heterozygous mutants phenotypes should be exacerbated compared to the homozygous mutant. This genetic approach will hopefully provide insight to how the conserved orc4^MGS mutation is functioning, which has the potential to be translated to humans.

Intestinal bacteria have been shown to be important in regulating drug metabolism of the host. However, little is known about the effect of exogenous bacteria, such as probiotic-treatment and conventionalization of germ-free conditions on the host hepatic drug-metabolizing enzymes and potential adverse “bug-drug” interactions. VSL#3 is a potent probiotic-combination that is clinically used for irritable bowel syndrome and ulcerative colitis. The purpose of this study was to quantify the expression of approximately 90 critical drug-metabolizing enzymes in livers of conventional and germ-free mice treated with or without VSL#3 (in drinking water for 4-weeks), as well as in livers of conventionalized (2-month) germ-free mice. Bacterial 16S rRNA qPCR in the large intestinal content confirmed the colonization of certain VSL#3 components. In liver, the cytochrome P450 (Cyp) 3a gene cluster, which encodes the major xenobiotic-metabolizing enzymes, as well as the Cyp4a and 4f gene clusters, which encode the lipid-metabolizing enzymes, were examined. RT-qPCR demonstrated that VSL#3 had minimal effects on the Cyp3a and 4a mRNAs; however, it increased the mRNAs of Cyp4f37 and Cyp4v3 in conventional mice. Germ-free condition decreased the mRNAs of Cyp3a11, 3a25, 3a41a/b, and 3a44, whereas conventionalization partially restored their expression. In contrast, germ-free condition increased the mRNAs of Cyp4a10, 4a14, 4a31, and 4a32, whereas conventionalization reduced their expression to conventional levels. Western blot confirmed that VSL#3 had no effect on Cyp3a11 or 4a14 proteins, whereas conventionalization restored the conventional levels of Cyp3a11 and further decreased Cyp4a14 protein. In conclusion, short-term VSL#3 has minor effects on the expression of hepatic drug-metabolizing enzymes; germ-free conditions markedly decreased the Cyp3a but increased the Cyp4a gene expression, whereas conventionalization normalizes the expression of these enzymes.

Hot flashes affect 75% of menopausal women, leaving them with recurring vasomotor symptoms such as temperature fluctuations, flushing, sweating, skin blotching, anxiety, heart palpitations, and sleep disturbances. Nearly 20% of these women describe their symptoms as “intolerable,” but unfortunately there are few safe options when treating hot flashes. Although hot flashes are associated with a decrease in circulating levels of sex hormones caused by ovarian aging and its effects on the brain, we do not understand the cellular and molecular mechanisms of the brain that actually trigger a hot flash. We hypothesize that the vasomotor symptoms of hot flashes originate from circuits in the hypothalamus that become hyper-activated by the abrupt decline in sex steroid production that typically occurs at menopause. As mice do not undergo menopause, our goal has been to develop a mouse model for hot flashes. To this end, we administered acyline, a GnRH antagonist that simulates menopause by suppressing sex steroid production. Concurrently, we measured core body temperature (CBT) with a tiny implantable recording device that recorded CBT every 30 seconds. This allowed us to look for changes in CBT that resembled menopausal hot flashes by comparing CBT patterns of experimental mice (acyline-treated) and controls (vehicle-treated). In hopes of identifying a CBT response to acyline, I developed computer software to analyze CBT data over time, isolate periods of interest, and compare temperature profiles between the experimental and control groups. My analysis revealed no clear effect of the GnRH antagonist on CBT in the mice, but did demonstrate clear diurnal changes in CBT reflecting normal activity patterns in rodents. Nevertheless, we remain optimistic that refinement of our experimental approach will yield an improved experimental model that simulates menopausal hot flashes.

Cancer patients in Washington State have been able to use medical marijuana (MJ) legally in consultation with health care professionals since 1998, but with the approval of I-502 in 2012, MJ availability and opportunities for use have increased in this diverse patient population. Limited medical literature, extensive lay press, and early data from local providers suggest that MJ use in cancer patients may be on the rise. However, smoking marijuana can present a major infectious risk, especially among patients whose disease or treatment compromises their immune system. Inhalation provides a perfect route for fungus and bacteria on the decaying plant to enter the lungs, and a number of studies have found fungal spores and bacterial growth on MJ leaves; others have associated MJ use with dangerous lung infections. Additional MJ products, such as baked goods and oils, have not been sufficiently studied in this population. The danger of infections in cancer patients provides an impetus to study the frequency of MJ use among this patient population. Through an evaluation of marijuana use at the Seattle Cancer Care Alliance (SCCA) we hope to more thoroughly describe how often both medical and recreational marijuana are used by cancer patients in the current era of legalization. Utilizing a patient survey developed in consort with Seattle King County Health Department and SCCA patients and staff, we will assess how and why patients use marijuana. The survey will collect information about demographics, frequency of use, methods for MJ delivery (e.g. edible vs. inhalation), influential factors on use (e.g. physician input vs. internet sources, blogs), and patient knowledge of the perceived dangers and benefits of MJ. Synthesis and analysis of survey data will provide the basis for education and future interventions for cancer patients considering MJ use.

Patients diagnosed with pulmonary hypertension (PHTN) are known to be at higher risk for post-operative complications. However, no studies have determined a correlation between PHTN and the severity of complications to date. While echocardiograms (ECHO) — the current gold standard for diagnosing both PHTN and its severity— are highly accurate, they are also expensive. However, the “conventional wisdom” within the Department of Anesthesiology at University of Washington Medical Center (UWMC) is that a functional status—measured in metabolic equivalence values, or METS— of ≥4 excludes the possibility of PHTN. We sought to determine if functional status could serve as a more cost-effective replacement for the traditional ECHO. Our study examined patients with PHTN who have undergone surgery at UWMC since 2007, stratified by severity. Our intern group abstracted data from medical records to collect pre-operative, peri-operative, and post-operative information. No significant correlation was found between METS and severity of PHTN, with a p-value of 0.979, thereby resolving the aforementioned misconception. The project scope was then redefined to look at the data with a focus on functional status. Most significantly, there was an increase seen in patients’ length of stay in those with a functional status of <4. Other correlations have been found, such as between functional status and with the American Society of Anesthesiologists class (IV or V >> II-III), or with gender (F>M). These correlations can serve to aid future research and direct medical advances towards becoming more beneficial and cost efficient for patients and healthcare professionals alike.

Juvenile idiopathic arthritis (JIA) is an inflammatory disease with no definite known cause and is one of the most common rheumatologic chronic conditions in children. An estimated 20% experience lifelong pain and significant disability. Complaints of sleep disturbances are common in JIA, and sleep disorders, such as sleep disordered breathing (SDB) may contribute to poor health outcomes in JIA. In a recent study from our laboratory, 40% of a sample of school-age children with JIA, regardless of disease status (e.g. active vs inactive disease), showed signs of SDB. SDB in children is a serious health concern associated with significant adverse health outcomes and high use of health care resources. SDB is a spectrum of pathologic disorders characterized by habitual snoring and repeated episodes of partial or complete upper airway obstruction during sleep that result in inadequate or fragmented sleep. An estimated 2-27% of school-age children likely have primary snoring, and 1-4% suffer from obstructive sleep apnea (OSA), but the proportion of children with JIA and SDB is unknown. The aims of this case control study were to compare the prevalence of SDB and SDB risk factors between JIA and control children; and among JIA children examine the risk factors for OSA. Children underwent one night of polysomnography in the UW School of Nursing Sleep Research Laboratory, a physical examination, and parents completed the Child Sleep Habits Questionnaire (CSHQ). Data were analyzed using SPSS. Analysis included: 1) examining group differences between JIA and control children in demographic, clinical characteristics, prevalence of SDB, and the prevalence of SDB risk factors; and 2) SDB risk factors among JIA children. Multiple comparisons were controlled for with a significance set at p <.01. Of the 30% of the sample who met clinical criteria for OSA, 81% had JIA; 10% were controls (p < 0.001).

Regional anesthesia via nerve block catheter placement in the brachial plexus is an effective means of post-operative analgesia for total shoulder replacement. Placement of a nerve block for patients undergoing such surgeries reduces opioid usage and the potential for associated potential side effects such as nausea, addiction, and diminishing results due to tolerance. However, due to brachial plexus blocks being in close proximity with the phrenic nerve (supplies diaphragm) patients experience reduced lung function from diffusion of the local anesthetic to the phrenic nerve. There are three common positions at which the nerve block catheter for shoulder surgery is placed: the suprascapular, supraclavicular, or interscalene positions, each showing efficacy in pain management, but with increasing proximities to the phrenic nerve. Traditionally, placement of the catheter for shoulder replacement has been at the interscalene level, due to the more distal blocks having higher risk of puncturing the lung itself and causing pneumothorax. With the increasing use of ultrasound to guide these procedures more accurately, additional blocks including the supraclavicular and suprascapular blocks have risen in popularity. We conducted a comparative study with 80 total shoulder replacement patients to determine superiority in pulmonary function between these three nerve blocks. Subjects were divided and received a continuous catheter ropivacaine nerve block at one of the aforementioned positions. Our study compared patients’ lung function via spirometry and ultrasound analysis of the diaphragm pre- and post-operation. Patient reported pain scores and opioid consumption over 24 hours were recorded to measure nerve block analgesia efficacy. We found that the cohort of more distal supraclavicular block patients had better lung function compared with the other two block placements cohorts with comparable pain control. By reducing the complications associated with regional anesthesia, we will improve patient comfort with invasive procedures and enhance patient recovery after surgery.